Anti-Fibronectin antibody [F14]

8 product images

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  Western blot - Anti-Fibronectin antibody [F14] (ab45688)

  Lane 1: Wild-type HAP1 whole cell lysate (20 μg)
  Lane 2: Fibronectin knockout HAP1 whole cell lysate (20 μg)
  

  Lanes 1 - 2: Merged signal (red and green). Green - ab45688 observed at 262 kDa. Red - loading control, ab18058, observed at 130 kDa.

  ab45688 was shown to react with Fibronectin in wild-type HAP1 cells as signal was lost in Fibronectin knockout cells. Wild-type and Fibronectin knockout samples were subjected to SDS-PAGE. ab45688 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/500 dilution and 1/2000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-Fibronectin antibody [F14] (ab45688)

  Predicted band size: 262 kDa

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  Flow Cytometry (Intracellular) - Anti-Fibronectin antibody [F14] (ab45688)

  Intracellular Flow Cytometry analysis of HepG2 (Human hepatocellular carcinoma epithelial cell) cells labeling Fibronectin with purified ab45688 at 1/150 dilution (10μg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).
  

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  Western blot - Anti-Fibronectin antibody [F14] (ab45688)

  Blocking and diluting buffer: 5% NFDM/TBST.

  All lanes: Western blot - Anti-Fibronectin antibody [F14] (ab45688) at 1/10000 dilution

  All lanes: Human serum lysates at 15 µg

  Secondary

  All lanes: Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

  Predicted band size: 262 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-Fibronectin antibody [F14] (ab45688)

  Immunocytochemistry/ Immunofluorescence analysis of HepG2 (Human hepatocellular carcinoma epithelial cell) cells labeling Fibronectin with Purified ab45688 at 1:500 dilution. Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

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  Western blot - Anti-Fibronectin antibody [F14] (ab45688)

  Blocking and diluting buffer: 5% NFDM/TBST

  All lanes: Western blot - Anti-Fibronectin antibody [F14] (ab45688) at 1/10000 dilution

  Lane 1: Mouse serum lysates at 15 µg

  Lane 2: Rat serum lysates at 15 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 262 kDa

  Observed band size: 263 kDa

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  Western blot - Anti-Fibronectin antibody [F14] (ab45688)

  All lanes: Western blot - Anti-Fibronectin antibody [F14] (ab45688) at 1/5000 dilution

  All lanes: human serum

  Predicted band size: 262 kDa

  Observed band size: 263 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-Fibronectin antibody [F14] (ab45688)

  ICC/IF image of unpurified ab45688 stained HepG2 (Human liver hepatocellular carcinoma cell line) cells. The cells were fixed in 4% formaldehyde (10 minutes) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab45688 at 1/250 dilution overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor^® 488 goat anti- rabbit (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1 hour. Alexa Fluor^® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43 μM for 1 hour at room temperature.

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  Western blot - Anti-Fibronectin antibody [F14] (ab45688)

  Western blot: Rabbit Monoclonal[F14] to Fibronectin ab45688 staining at 1/1000 dilution, shown in green; Mouse anti-CANX (Anti-Calnexin antibody [CANX/1543] ab238078) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 238-268 kDa in Wild-type MCF7 cell lysates with no signal observed at this size in FN1 knockout MCF7 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

  All lanes: Western blot - Anti-Fibronectin antibody [F14] (ab45688) at 1/1000 dilution

  Lane 1: Wild-type MCF7 at 20 µg

  Lane 2: Western blot - Human FN1 knockout MCF7 cell line (Human FN1 knockout MCF7 cell line ab286324) at 20 µg

  Lane 3: HepG2 at 20 µg

  Lane 4: PC-3 at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 272 kDa

  Observed band size: 238 kDa, 268 kDa